The present invention relates generally to methods and apparatus for delivery of macromolecules into cells. More specifically, the present invention provides methods and apparatus for delivering substances, such as macromolecules, e. g. deep tumor tissue treating agents, polynucleotide vaccines (DNA vaccine and/or RNA vaccine) and protein-based vaccines, into cells in tissues.
The first DNA vaccination procedure in the prior art was called naked DNA vaccination because a liquid solution of DNA was injected into the muscle of mice with no additives to enhance transfection. This method does transfect a few cells and does induce an immune response to the expressed antigen in mice. However, in humans and primates, the method does not work well.
In the prior art, an improvement in DNA vaccine efficiency was obtained by the use of a biolistic method for DNA delivery. The biolistic method is done by coating metal microbeads with DNA and shooting the particles into skin after accelerating the particles to a chosen velocity. This method works much better than naked DNA. Part of the reason is that the DNA coated particles are injected into the skin to a depth that increases the chance of transfecting Langerhans cells. However, the biolistic method has some disadvantages. First, it causes some skin damage that may scar in some individuals. Second, in spite of the increased efficiency, more efficiency is needed. Third, the ballistic particle remains inside the patient after treatment. In this respect, it would be desirable if a method for delivering DNA to biological cells were provided which does not cause skin damage that results in scarring. Also, it would be desirable if a method for delivering DNA to biological cells were provided which does not leave a residue of ballistic particles in cells that are treated. As a matter of interest, the following U.S. patents disclose biolistic methods: U.S. Pat. Nos. 5,036,006 and 5,478,744.
A number of additional approaches to delivering macromolecules to biological cells are disclosed in the prior art and are represented by the following U.S. patents or other publications as follows.
U.S. Pat. No. 5,019,034 of Weaver et al discloses a process for electroporation of tissues in which electrodes are placed on top of the tissue surface, such as skin, of a patient. Molecules that are used for treating the skin are placed in reservoirs on top of the skin surface, and the treatment molecules must penetrate into the skin tissues transdermally. That is, the treatment molecules must pass from outside the skin to inside the skin. Not only is the surface layer of the skin relatively impermeable, if the layers of the skin to be treated are near the basal lamina of the epidermis, then the treatment molecules must traverse considerable skin tissue before the cells to be treated are reached by the treatment molecules. Such a treatment method is inefficient for treating cells near the basal lamina. Rather than using electrodes that are placed on the skin surface and have treatment molecules pass through the skin transdermally to treat biological cells near the basal lamina of the epidermis, it would be desirable if an electroporation method were provided for delivering molecules to biological cells in the epidermis, near the basal lamina, without having the treatment molecules pass through the skin transdermally.
U.S. Pat. No. 5,273,525 of Hofmann discloses an apparatus for electroporation of drugs and genetic material into tissues which employs relatively long hollow hypodermic needle for placing the drugs and genetic material in the vicinity of the tissues to be electroporated. Whenever a hollow hypodermic is employed in a tissue, the tissue is cut with a circular cut by the hollow hypodermic needle. As a result, when a patient receives hypodermic injection, the patient has considerable pain. To avoid such a circular cut, and to avoid the considerable pain involved, it would be desirable if a method for delivering molecules to biological cells were provided which does not employ a hypodermic needle.
U.S. Pat. No. 5,318,514 of Hofmann discloses an applicator for the electroporation of drugs and genes into cells. The applicator includes a plurality of needle electrodes which can be penetrated into the skin of a patient. Material to be electroporated into the skin is retained in a fluid reservoir which wets an open cell foam elastomer carrier for the fluid. Because the material to be electroporated is retained in a fluid, in both the reservoir and the open cell foam elastomer, careful control of the amount of the material at the electrode surfaces is difficult. It is difficult to control how much fluid flows down from the reservoir and the open cell foam elastomer to the surfaces of the needle electrodes, and, thereby, it is difficult to control how much of the treatment molecules is actually present on the surfaces of the electrodes 16 as the electroporation process is being carried out on the patient. Moreover, the presence of the fluid medium can have a flushing or washing effect on the tissues that are electroporated in such a way that the electroporation process is interfered with. In these respects, it would be desirable if an electroporation method for delivering molecules to biological cells were provided which does not employ a fluid medium that flows down onto the electrodes as the electroporation process is being carried out on the patient.
Other disclosures relating to the use of electroporation to mediate gene transfer into epidermal cells are found in an article by Reiss et al entitled xe2x80x9cDNA-mediated gene transfer into epidermal cells using electroporationxe2x80x9d in Biochem. Biophys. Res. Commun., Vol. 137, No. 1, (1986), pages 244-249 and in an article by Titomirov entitled xe2x80x9cIn vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNAxe2x80x9d in Biochim. Biophys. Acta., Vol. 1088, No. 1, (1991), pages 131-134.
U.S. Pat. No. 5,389,069 of Weaver discloses a method and apparatus for in vivo electroporation of tissues which employs a relatively long hollow cylindrical needle for providing treating substances deep into tissues. As mentioned above, avoiding the use hollow cylindrical needles would be desirable to avoid the pain involved therewith.
U.S. Pat. Nos. 5,580,859 and 5,589,466, both of Felgner et al, disclose a method of delivering macromolecules into muscles and skin of a patient by an injection method. Their method does not employ electroporation.
U.S. Pat. No. 5,697,901 of Eriksson discloses gene delivery into tissues by the use of a gene-carrying fluid medium that is pressurized in conjunction with hollow microneedles. Problems of control and flushing using fluid media have been discussed hereinabove. An electroporation step is not employed in the Eriksson patent. As a matter of interest, Eriksson addresses the subject of pain in two respects. There is a statement that the hollow microneedle system can be used for treating pain. There is a statement that pain in wounds can be relieved by cooling. It is noted by the present inventors herein that Eriksson does not discuss his treatment method per se as being of a pain free or reduced pain treatment method. The present inventors theorize that the pressurized fluid injection method that is employed by Eriksson is not conducive to a pain free or reduced pain treatment method. In this respect, it would be desirable to provide a gene therapy treatment method that employs micro-sized needles, but that does not employ a pressurized fluid injection step for injecting fluid into a patient.
In an article by Henry et al entitled xe2x80x9cMicrofabricated Microneedles: A Novel Approach to Transdermal Drug Deliveryxe2x80x9d in Journal of Pharmaceutical Sciences, Vol. 87, No. 8, August 1998, pages 922-925, there is a disclosure that an array of microneedles are employed to penetrate the epidermis to leave micro-sized perforations to facilitate transdermal permeability of fluid-carried treatment agents into the microperforated epidermis. Because the microneedles are inserted only a microscopic distance into the epidermis, use of the microneedles is potentially nonpainful. There is no disclosure that the microneedles are to be used as electrodes. Also, an electroporation step is not disclosed in the Henry et al article.
The following U.S. patents may be of interest for their disclosure of the use of relatively long electrodes in treating biological cells: U.S. Pat. No. 5,439,440 of Hofmann; U.S. Pat. No. 5,468,223 of Mir; U.S. Pat. No. 5,674,267 of Mir et al; U.S. Pat. No. 5,702,359 of Hofmann et al; U.S. Pat. No. 5,810,762 of Hofmann; and U.S Pat. No. 5,873,849 of Bernard. It is noted that none of the patents listed in this paragraph disclose the use of relatively long electrodes which have fixed electrode surfaces which are coated with a static layer of electrode releasable molecules for treating the biological cells either when an electric field is applied to the electrodes or when the static layer dissolves off of the electrodes in a solvent near the biological cells.
Further with respect to the issue of reduced pain treatment, it is noted that two important electrical parameters in electroporation are closely related to a perceived pain in vivo. One parameter is absolute voltage experienced by the in vivo tissue. Another parameter is the pulse width experienced by the in vivo tissue. In these respects, it would be desirable to provide an electroporation method for delivering molecules to biological cells which applies relatively low absolute voltage to cells undergoing electroporation and which can be used, if desired, to apply pulses having relatively short pulse width to the cells undergoing electroporation.
Still other features would be desirable in a method and apparatus for delivery of macromolecules into epidermal cells. For example, when electrodes are penetrated into the epidermis, the conductive base electrode portions and the conductive tips of the electrodes may exhibit electrical characteristics which are undesirable with respect to the electroporation process in general and the biological cells that are treated in particular. In this respect, it would be desirable if a method and apparatus for delivery of macromolecules into epidermal cells were provided which render nonconductive the base portions and tip portions of the electrodes.
Once electrode assemblies having a plurality of needle electrodes have been employed on a patient, it may be a difficult task to clean and sterilize them for a subsequent use. In this respect, it would be desirable if a method and apparatus for delivery of macromolecules into cells were provided in which the electrode assemblies are disposable.
When disposable electrode assemblies are employed, it would be desirable if the disposable electrode assemblies are packaged in sterile packaging.
Thus, while the foregoing body of prior art indicates it to be well known to use electroporation to deliver molecules to biological cells, the prior art described above does not teach or suggest a method and apparatus for delivery of macromolecules into cells which has most of the following combination of desirable features: (1) does not cause skin damage that results in scarring; (2) does not leave a residue of ballistic particles in cells that are treated; (3) provides an electroporation method for delivering molecules to biological cells in the epidermis, near the basal lamina, without having the treatment molecules pass through the skin transdermally; (4) does not employ a hypodermic needle; (5) does not employ a fluid medium that flows down onto the electrodes as the electroporation process is being carried out on the patient; (6) does not employ a pressurized fluid injection step for injecting fluid into a patient; (7) applies relatively low absolute voltage to cells undergoing electroporation; (8) if desired, can be used to apply pulses having relatively short pulse width to the cells undergoing electroporation; (9) renders the base portions and tip portions of the electrodes nonconductive; (10) provides disposable electrode assemblies; (11) provides electrode assemblies which are packaged in sterile packaging: and (12) permits treatment of tissues using coated long electrodes which have electrode releasable material which includes a tissue treating agent. The foregoing desired characteristics are provided by the unique electrodes coated with treating agent and uses thereof, of the present invention as will be made apparent from the following description thereof. Other advantages of the present invention over the prior art also will be rendered evident.
It is noted that aspects of the invention have been disclosed in copending PCT International Application No. PCT/US00/00014, filed Jan. 12, 2000, which is based upon copending U.S. Provisional Application Ser. No. 60/117,755, filed Jan. 28, 1999. The PCT International Application No. PCT/US00/00014 was published on Aug. 3, 2000 with PCT International Publication Number WO 00/44438. In addition to currently disclosing some of those aspects of the invention previously disclosed in the above-mentioned PCT and U.S. Provisional applications, the present application discloses additional invention aspects.
In accordance with one aspect of the invention, a method is provided for delivery of molecules into biological cells which includes the steps of:
(a) providing electrodes in an electrode assembly, wherein the electrodes have a fixed electrode surface,
(b) coating the fixed electrode surfaces of the electrodes with at least one static layer of electrode releasable molecules to be delivered,
(c) attaching the electrode assembly having the statically coated electrodes to an electrode assembly holder,
(d) providing a waveform generator for generating electric fields,
(e) establishing electrically conductive pathways between the electrodes and the waveform generator,
(f) locating the electrodes such that the biological cells are situated therebetween, and
(g) providing electric fields in the form of pulse waveforms from the waveform generator to the electrodes, such that molecules in the at least one static layer of the electrode releasable molecules on the electrodes are delivered into the biological cells.
On the one hand, when the static layer of electrode releasable molecules does not include solvent separable material, then substantially all of the static layer of electrode releasable molecules are electric field separable molecules. In such a case, the electric field separable molecules are both driven off of the electrodes and delivered into the biological cells by the applied electric fields.
On the other hand, when the static layer of electrode releasable molecules does include solvent separable material, such as solvent separable solid material, then the static layer of electrode releasable molecules includes both solvent separable solid material and electric field separable molecules. In such a case, a solvent dissolves the solvent separable material thereby releasing the electric field separable molecules from the electrode, and the electric field separable molecules are delivered into the biological cells by the applied electric fields. The solvent includes body fluids which are present in body tissues.
Often, the electrode releasable molecules are in a form of a static coating on the fixed electrode surface. In this respect, the term xe2x80x9cstaticxe2x80x9d means that the coating remains stationary on the fixed electrode surface when either not in tissues or not under the influence of an electric field. However, such a static coating moves off of the fixed electrode surface either when it is dissolved off of the fixed electrode surface or when it is driven off of the fixed electrode surface under the influence of either a solvent or a suitable electric field, respectively.
A number of benefits can be realized by employing the static coated electrodes of the present invention. For example, a pre-measured quantity of a static layer of electrode releasable molecules can be retained on the fixed electrode surfaces. Such a pre-measured quantity of the static layer of electrode releasable molecules can serve as a pre-measured dose of material to be delivered to the biological cells. Moreover, the static coated electrodes can be coated with a concentrated quantity of the electrode releasable molecules. In addition, the static coated electrodes can be pre-packaged so that when they are removed from their package, they are rapidly ready for use, without the need for conventional preparatory steps such as dilution and hypodermic injection.
Also, in accordance with aspects of the present invention, an electrode includes an electrode underbody and a fixed electrode surface which lies on top of the electrode underbody. The fixed electrode surface can be implemented in a wide variety of embodiments. For example, most simply, the simple surface of the electrode itself can serve as the fixed electrode surface which lies on top of the electrode underbody. The fixed electrode surface can be a smooth electrode surface, can be an oxidized metal surface (e. g. oxides of silver, nickel, and copper), can include fixed metal particles, and can be a roughened surface.
Also, in accordance with aspects of the present invention, the electrode releasable material on the fixed electrode surface can be in a form of a gel coating, a solid layer of nonpolymeric material, and a polymer layer.
Varieties of the fixed electrode surface and the static, electrode releasable material can be mixed and matched.
Some specific examples of combinations of the fixed electrode surface and the electrode releasable material include: a fixed electrode surface having metal oxides and electrode releasable material including DNA; a fixed electrode surface being a smooth surface and the electrode releasable material in the form of a solid coating; a fixed electrode surface having an etched rough surface and the electrode releasable material as either a solid nonpolymeric layer, a gel layer, or a polymeric layer.
The pulse waveforms may be provided by applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, to the biological cells. The sequence of at least three DC electrical pulses has one, two, or three of the following characteristics (a) at least two of the at least three pulses differ from each other in pulse amplitude, (b) at least two of the at least three pulses differ from each other in pulse width, and (c) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses.
Additionally, the method can include a step of providing the electrode assembly holder with electrically conductive pathways between the electrode assembly and the waveform generator.
In addition, the method can include a step of providing the electrode assembly in a sterile package. In such a case, the electrode assembly is removed from the sterile package prior to use.
Further, the method can include the steps of providing the electrodes with electrically insulated outer surface electrode tip portions and electrically insulated outer surface electrode base portions.
The molecules in the at least one static layer of molecules in the electrode coating preferably include macromolecules. The macromolecules in the electrode coating can include a tissue treating agent, a polynucleotide vaccine (DNA vaccine and/or RNA vaccine), or a protein-based vaccine, among others.
With a variation of the method of the invention, the molecules can be delivered to Langerhans cells in epidermal tissue of a patient with reduced sensation (reduced pain or nearly painless or pain free) to the patient. To provide reduced sensation delivery of molecules to the patient, the following conditions are maintained (a) the pulse waveforms have an absolute applied voltage in a range of 0.1 to 300 volts; (b) the electrodes of opposite polarity are separated by a separation distance in a range of from 50 to 500 microns; and (c) the electrodes are penetrated into the epidermal tissue a distance up to and slightly beyond the basal lamina layer of the epidermal tissue.
With another variation of the method of the invention, the molecules can be delivered to a tissue which is deeply located under healthy tissue. With such a variation of the method of the invention, the electrodes are long enough to penetrate through the healthy tissue and into the tumor. The fixed electrode surface portions of the electrodes that penetrate the tumor are coated with electrode releasable material that includes a deep tumor tissue treating agent.
The pulse waveforms which drive the molecules of the electrode releasable coating molecules off of the electrodes are electrophoresis waveforms. The pulse waveforms which deliver the driven-off molecules into the biological cells are electroporation waveforms. For a static layer of electric field separable molecules, common pulse waveforms both drive the coating molecules off of the electrodes and deliver the driven-off molecules into the biological cells.
The biological cells can be in vivo, ex vivo, or in vitro. More specifically, the biological cells can be in epidermal tissue and can be Langerhans cells in the epidermal tissue. Also, the biological cells can be in deep tissues, and can be in tumors in deep tissues.
In accordance with another aspect of the invention, an apparatus is provided for delivery of molecules into biological cells and includes a waveform generator which provides pulse waveforms. An electrode assembly holder is provided, and an electrode assembly is mechanically supported by the electrode assembly holder. The electrode assembly holder is also electrically connected to the waveform generator through electrically conductive pathways. The electrode assembly includes electrodes which are coated with at least one static layer of molecules to be delivered into the biological cells.
The electrode assembly can be removable and replaceable from the electrode assembly holder. In this respect, the electrode assembly includes electrode-assembly-conductive strips. The electrode assembly holder includes holder conductors which are registrable with the electrode-assembly-conductive strips when the electrode assembly is mechanically connected to the electrode assembly holder. Also, the electrode assembly holder includes electrically conductive pathways between the holder conductors and the waveform generator.
With the apparatus, a sterile package can be provided for the electrode assembly. The sterile package is removed from the electrode assembly after the electrode assembly is mechanically supported by the electrode assembly holder and is electrically connected to the waveform generator.
With the apparatus, if desired, the waveform generator provides pulse waveforms which include a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, to the biological cells. The sequence of at least three DC electrical pulses has one, two, or three of the following characteristics (a) at least two of the at least three pulses differ from each other in pulse amplitude, (b) at least two of the at least three pulses differ from each other in pulse width, and (c) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses.
The electrodes can include electrically insulated outer surface electrode tip portions and electrically insulated outer surface electrode base portions. The electrodes are coated with at least one static layer of molecules, which may include macromolecules, which may include a tissue treating agent, a polynucleotide vaccine (a DNA vaccine and/or a RNA vaccine) and/or a protein-based vaccine.
The static layer of electrode releasable macromolecules on the electrodes (e. g. a tissue treating agent, a polynucleotide vaccine, or a protein-based vaccine, among others) can be in a variety of forms prior to using the electrodes on a patient. More specifically, the static layer of macromolecules can be in a solid form, coating the solid electrodes. Also, the static layer of macromolecules can be in a gel form or can be in a form of a liquid fixed on a fixed surface matrix of the electrodes. The fixed surface matrix can be solid surface particles (e. g. metal particles), a liposome matrix, or a solid polymer matrix.
In accordance with yet another aspect of the invention, a packaged sterile electrode assembly is provided which includes a sterile electrode assembly which includes electrodes which are coated with at least one static layer of molecules to be delivered into biological cells. The electrode assembly includes electrode-assembly-conductive strips for connection to complementary electrically conductive pathways leading to the waveform generator. In addition, an internally sterile package encloses the sterile electrode assembly contained therein.
With the packaged sterile electrode assembly, the electrodes can include electrically insulated outer surface electrode tip portions and electrically insulated outer surface electrode base portions.
With the packaged sterile electrode assembly, the electrodes are coated with macromolecules which can include a solid phase polynucleotide (DNA vaccine and/or RNA vaccine) and/or a solid phase protein-based vaccine. Also, the polynucleotide vaccine or protein-based vaccine can be in a gel form or can be in a form of a liquid fixed on a fixed surface matrix of the electrodes, prior to using the electrodes on a patient. The fixed surface matrix can be solid surface particles (e. g. metal particles), a liposome matrix, or a solid polymer matrix.
In accordance with the invention, transfection of cells with DNA in vivo, using electric field mediated transfection, is an efficient process. Additionally, electric fields can be used for the delivery of other macromolecules such as RNA and proteins into cells. In the prior art, the electric field delivery has one disadvantage, that being the pain induced by the high voltage electrical pulses required for the transfection. In contrast, as described herein, a method is provided for delivering macromolecules (DNA, RNA, and protein) to cells, in tissues in vivo, using painless (or nearly painless) and efficient electric field mediated delivery.
A number of applications of the method and apparatus for delivery of macromolecules into cells, of the invention, are contemplated. Briefly, such applications include treating organ tissues, polynucleotide vaccination, protein vaccination, and gene therapy
For treating deep tumor tissues, it is important to maximize delivery of the deep tumor tissue treating agent to the tumor tissue and to minimize deliver of the agent to healthy tissue.
For DNA vaccination, there are two overriding requirements. One is gene expression in vivo and the other is that antigen-presenting cells must either obtain antigen from a nearby, transfected cell or express the antigen themselves. The highest concentration of accessible antigen presenting cells resides in the skin as cells called Langerhans cells. These cells are part of a very effective group of antigen presenting cells called dendritic cells. Electroporation is a viable alternative method for transfecting selected cells in vivo.
Proteins also can be introduced into cells using electric field mediated delivery. In conventional vaccination, proteins are delivered outside cells using a hypodermic needle. This type of delivery is inefficient in inducing a cell mediated cytotoxic lymphocyte immune response. Some infectious diseases require a cytotoxic lymphocyte response as a component of the immune response for efficient clearance of the infection. Delivery of proteins into cells promotes the induction of that response.
Delivery of therapeutic genetic medicine into cells for the purpose of making those cells express a missing protein is the basis of gene therapy. Using relatively short electrodes of the invention, the method and apparatus of the invention can be used to deliver therapeutic DNA into cells on the surface of any accessible organ in addition to the skin. Using relatively long electrodes of the invention, the method and apparatus of the invention can be used to deliver therapeutic DNA into cells deep into tissues and organs.
The method of the invention can be used in a method for painless, effective delivery of macromolecules to epidermal tissues, in vivo, for the purpose of vaccination (or treatment), DNA vaccination, gene therapy, or other reasons.
An electrode with at least one of two characteristics is used for delivery of macromolecules into cells in epidermal tissue. One of the two characteristics is an electrode length short enough that it does not penetrate to a depth in tissue with nerve endings. Another characteristic is that inter-electrode distances are small enough to allow pulse parameters (voltage and pulse width) to be used that are painless. Only one or the other of these characteristics is needed in any given epidermal application, however, they may be used together.
The above brief description sets forth rather broadly the more important features of the present invention in order that the detailed description thereof that follows may be better understood, and in order that the present contributions to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will be for the subject matter of the claims appended hereto.
In this respect, before explaining preferred embodiments of the invention in detail, it is understood that the invention is not limited in its application to the details of the construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood, that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception, upon which disclosure is based, may readily be utilized as a basis for designing other structures, methods, and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
In view of the above, it is an object of the present invention to provide a new and improved method and apparatus for delivery of macromolecules into cells which does not cause skin damage that results in scarring.
Another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which does not leave a residue of ballistic particles in cells that are treated.
Even another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that provides an electroporation method for delivering molecules to biological cells in the epidermis, near the basal lamina, without having the treatment molecules pass through the skin transdermally.
Still a further object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which does not employ a hypodermic needle.
Yet another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that does not employ a fluid medium that flows down onto the electrodes as the electroporation process is being carried out on the patient.
Still another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which does not employ a pressurized fluid injection step for injecting fluid into a patient.
Yet another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that applies relatively low absolute voltage to cells undergoing electroporation.
Still a further object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that can be used, if desired, to apply pulses having relatively short pulse width to the cells undergoing electroporation.
Yet another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which renders the base portions and tip portions of the electrodes nonconductive.
Still a further object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that provides disposable electrode assemblies.
Yet another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which electrode assemblies are packaged in sterile packaging.
Still another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which permit treatment of tissues using coated long electrodes which have electric field separable material which includes a tissue treating agent.
These together with still other objects of the invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be had to the accompanying drawings and descriptive matter in which there are illustrated preferred embodiments of the invention.